Large liposome composition not increasing ldl levels and method of treating atherosclerosis related thereto

ABSTRACT

The present invention provides a pharmaceutical composition consisting essentially of large liposomes comprised of phospholipids substantially free of sterol. The composition forces the reverse transport of cholesterol from peripheral tissues to the liver in vivo. The invention further provides a method of treating atherosclerosis in a subject comprising the step of administering a liposome composition to the subject. The liposome composition is selected from the group consisting of unilamellar liposomes and multilamellar liposomes and the liposomes have an average diameter of about 50-150 nanometers. LDL levels in said subject do not increase with utilization of the method. The invention also provides a method of controlling cholesterol metabolism in hepatic parenchymal cells in a subject in vivo through cell-cell communication from Kupffer cells to the parenchymal cells. The method includes the step of administering a liposome composition to a subject. The liposome composition is selected from the group consisting of large unilamellar liposomes and large multilamellar liposomes, and the liposomes having an average diameter of about 50-150 nanometers. Similarly, LDL levels in the subject do not increase. In variants, the liposome composition is given periodically, given more than once, or given in repeated doses. The liposomes have diameters larger than about 50 nm, diameters larger than about 80 nm, and diameters larger than about 100 nm in different variants. The liposomes are phospholipids selected from the group consisting of phosphatidyl choline, phosphatidyl glycerol, palmitoyl-oleoyl phosphatidyl choline, combinations thereof, and derivatives thereof.

CONTINUING DATA

[0001] This application is a continuation in part regular patentapplication of pending U.S. provisional patent application serial No.60/005,090 filed by Kevin Jon Williams, a citizen of the United States,residing at 425 Wister Road, Wynnewood, Pennsylvania 19096 on Oct. 11,1995 entitled “METHOD OF FORCING THE REVERSE TRANSPORT OF CHOLESTEROLFROM PERIPHERAL TISSUES TO THE LIVER IN VIVO WHILE CONTROLLING PLASMALDL AND COMPOSITIONS THEREFOR.” Pending U.S. provisional patentapplication serial No. 60/005,090 filed Oct. 11, 1995 is attached to theinstant regular patent application as attachment A. Applicant expresslyincorporates attachment A hereto into the instant regular patentapplication by reference thereto as if fully set forth.

BACKGROUND OF THE INVENTION

[0002] Atherosclerosis, a major killer in Western countries, ischaracterized by the accumulation of cholesterol and cholesteryl esterin cells and in extracellular areas of the arterial wall and elsewhere.These lipids have potentially harmful biologic effects, for example, bychanging cellular functions and by narrowing the vessel lumen,obstructing the flow of blood. Removal of these lipids would providenumerous, substantial benefits. A major obstacle to removing excessarterial cholesterol in vivo has been disposition of the cholesterolmobilized from tissues, cells, extracellular areas, and membranes.Natural (e.g., high-density lipoprotein) and synthetic (e.g., smallliposomes) particles that could mobilize peripheral tissue lipids have asubstantial disadvantage: they deliver their lipids to the liver in amanner that disturbs hepatic cholesterol homeostatis and to an incorrectpool in the liver, resulting in elevations in plasma concentrations ofharmful lipoprotein, such as low-density lipoprotein (LDL), a majoratherogenic lipoprotein. There exists a need for better methods tomanipulate the lipid content and composition of peripheral tissues,cells, membranes, and extracellular regions in vivo.

[0003] The intravenous administration of cholesterol-poor phospholipidvesicles (liposomes) or other particles to transport cholesterol andother exchangeable material from lipoprotein and peripheral issues,including atherosclerotic arterial lesions, to the liver producessubstantial derangements in hepatic cholesterol homeostasis, such asenhanced hepatic secretion of apolipoprotein-B, and suppression ofhepatic LDL receptors. The hepatic derangements lead to increase plasmaconcentrations of LDL and other atherogenic lipoprotein. Increasedconcentrations of LDL or other atherogenic lipoprotein will accelerate,not retard, the development of vascular complications. There exists aneed for methods and compounds that can produce removal of cholesterolfrom cellular and extracellular regions of arteries, but withoutprovoking a rise in the plasma concentration of LDL.

[0004] The invention described herein provides methods and compositionsrelated to the removal of cholesterol from arteries, whole controllingplasma concentrations of LDL. The present invention addresses theseneeds so that diseases and detrimental medical conditions can betreated, controlled or eliminated. The present invention targets amarket of tens of millions of individuals world-wide who suffer frommedical conditions the present invention is directed to solving.

[0005] This invention methods and compositions that related to the“reverse” transport of lipids and other exchangeable material fromperipheral tissues to the liver in vivo while controlling plasma LDLconcentrations. There exists a need for a method of, treatment, and apharmaceutical composition for forcing the reverse transport of lipidsfrom peripheral tissues to the liver in vivo while controlling plasmaLDL concentrations; of regulating hepatic parenchymal cell cholesterolcontent and metabolism in a cell having at least one gene selected fromthe group consisting of a gene for an LDL receptor, a gene for HMG-CoAreductase, a gene for cholesterol 7-alpha-hydroxylase, and a generegulating a function involved in cholesterol homeostasis; and,homeostasis thereof; suppressing hepatic expression of a cholesterolester transfer protein gene in vivo, whereby plasma LDL and HDL arecontrolled as a result of said administration; suppressing the rise inplasma LDL concentrations after administration of an agent having smallacceptors of cholesterol or other lipids; of diagnosing a side-effect ofreverse transport of cholesterol from peripheral tissues to the liver invivo accompanying parenteral administration of a multiplicity of largeliposomes and small liposomes during a treatment period, whereby a sideeffect of administration of said liposomes is diagnosed and effectivelyregulated; and, diagnosing and treating a side-effect of reversetransport of lipids from peripheral tissues to the liver in vivoaccompanying parenteral administration of a multiplicity of largeliposomes and small liposomes during a treatment period. There furtherexists a need for a system in which patients will have a decreased riskof developing atherosclerosis and/or cellular changes from aging; animproved method of reducing the lipid content of lesions. Other needsand solutions to these needs will be revealed further herein.

SUMMARY OF THE INVENTION

[0006] The present invention provides a pharmaceutical compositionconsisting essentially of large liposomes comprised of phospholipidssubstantially free of sterol. The composition forces the reversetransport of cholesterol from peripheral tissues to the liver in vivo.The invention further provides a method of treating atherosclerosis in asubject comprising the step of administering a liposome composition tothe subject. The liposome composition is selected from the groupconsisting of unilamellar liposomes and multilamellar liposomes and theliposomes have an average diameter of about 50-150 nanometers. LDLlevels in said subject do not increase with utilization of the method.

[0007] The invention also provides a method of controlling cholesterolmetabolism in hepatic parenchymal cells in a subject in vivo throughcell-cell communication from Kupffer cells to the parenchymal cells. Themethod includes the step of administering a liposome composition to asubject. The liposome composition is selected from the group consistingof large unilamellar liposomes and large multilamellar liposomes, andthe liposomes having an average diameter of about 50-150 nanometers.Similarly, LDL levels in the subject do not increase. In variants, theliposome composition is given periodically, given more than once, orgiven in repeated doses.

[0008] The liposomes have diameters larger than about 50 nm, diameterslarger than about 80 nm, and diameters larger than about 100 nm indifferent variants. Administration is selected from the group ofparenteral administration, intravenous administration, intra-arterialadministration, intramuscular administration, subcutaneousadministration, transdermal administration, intraperitonealadministration, intrathecal administration, via lymphatics,intravascular administration, including administration into capillariesand arteriovenous shunts, rectal administration, administration via achronically indwelling catheter, and administration via an acutelyplaced catheter, and given in about 10 to about 1600 mg/kg/dose of theliposome composition. The liposomes are phospholipids selected from thegroup consisting of phosphatidyl choline, phosphatidyl glycerol,palmitoyl-oleoyl phosphatidyl choline, combinations thereof, andderivatives thereof.

[0009] It is an object of the invention to provide for better methods tomanipulate the lipid content and composition of peripheral tissues,cells, membranes, and extracellular regions in vivo.

[0010] It is a further object of the invention to provide for methodsand compounds that can produce removal of cholesterol from cellular andextracellular regions of arteries, but without provoking a rise in theplasma concentration of LDL.

[0011] The objects and features of the present invention, other thanthose specifically set forth above, will become apparent in the detaileddescription of the invention set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a side cross-sectional view of a lipoprotein and aliposome;

[0013]FIG. 2 illustrates a table of hepatic mRNA content (pg/μg) forCETP, HMG-CoAR, LDL receptors, and 7a-hydroxylase; and LDL ChE;

[0014]FIGS. 3 and 4 illustrate plasma LDL cholesteryl esterconcentrations in response to injections of LUVs, SUVs or saline overtime in one variant;

[0015]FIG. 5 illustrates LDL receptor mRNA levels in liver in responseto injections of LUVs, SUVs or saline over time;

[0016]FIG. 6 illustrates HMG-CoA reductase mRNA levels in liver inresponse to injection of LUVs, SUVs, or saline;

[0017]FIG. 7 Illustrates cholesteryl ester transfer protein mRNA levelsin liver in response to injection of LUVs, SUVs, or saline;

[0018]FIG. 8 illustrates 7-alpha hydroxylase mRNA levels in liver inresponse to injections of LUVs, SUVs, or saline;

[0019]FIG. 9 illustrates key points about LUVs and atherosclerosis;

[0020]FIG. 10 illustrates plasma LDL unesterified cholesterolconcentrations in response to injections of LUVs, SUVs or saline overtime;

[0021]FIG. 11 illustrates plasma LDL esterified cholesterolconcentrations in response to injections of LUVs, SUVs or saline overtime;

[0022]FIG. 12 illustrates LDL esterified cholesterol concentrations inresponse to injections of LUVs, SUVs or saline;

[0023]FIG. 13 illustrates plasma VLDL esterified cholesterolconcentrations in response to injections of LUVs, SUVs or saline ;

[0024]FIGS. 14 and 15 illustrate HDL esterified cholesterolconcentrations in response to injections of LUVs, SUVs or saline;

[0025]FIG. 16 illustrates the time course of cholesterol mobilizationfollowing an LUV injection into control or apoe KO m ice;

[0026]FIG. 17 illustrates the time course of LUV clearance in controlmice and apoE mice;

[0027]FIG. 18 illustrates that the compositions and methods of thepresent invention are effective in humans;

[0028]FIG. 19 illustrates a perspective view of an improved hemodialysissystem of the present invention and improved method of hemodialysis;

[0029]FIG. 20 illustrates a perspective view of an improved peritonealdialysis system 2000 and method of peritoneal dialysis;

[0030]FIG. 21 illustrates a perspective view of a variant of an improvedperitoneal dialysis system with assaying means 2100 and method ofperitoneal dialysis and analysis of spent fluid;

[0031]FIG. 22 illustrates a perspective view of an improved cardiaccatheterization and/or angioplasty system 2200 and method of cardiaccatheterization and/or angioplasty;

[0032]FIG. 23 illustrates a perspective view of a variant of an improvedcardiac catheterization and/or angioplasty system 2300 and method ofcardiac catheterization and/or angioplasty;

[0033]FIG. 24 illustrates a graph of hepatic lipid contents in responseto injections of LUVs, SUVs, or saline;

[0034]FIG. 25 illustrates plasma free cholesterol concentrationsfollowing repeated injections of SUVs or LUV (300 mg/kg) in NZW rabbits;

[0035]FIG. 26 illustrates plasma cholesterol ester concentrationsfollowing repeated injections of SUVs or LUV (300 mg/kg) in NZW rabbits;

[0036]FIG. 27 illustrates alternations in plasma components afterrepeated injections of SUVs; and,

[0037]FIG. 28 illustrates an agarose gel electrophoresis of whole plasmafollowing repeated injections of LUVs, SUVs, or saline.

DETAILED DESCRIPTION OF THE INVENTION

[0038]FIG. 1 illustrates a schematic illustration of the structure of anormal lipoprotein 100 and a unilamellar liposome 200. Lipoprotein 100and liposome 200 are comprised of a phospholipid molecule 300.Phospholipid molecules generally have polar head 500 and a fatty acylchains 400. Molecule 600 represents a molecule of unesterifedcholesterol. Lipoprotein 100 is comprised of a hydrophobic core 102composed mainly of triglycerides and cholesteryl esters surrounded by amonolayer of phospholipid molecules 300 with their fatty acyl sidechains 400 facing the hydrophobic core 102 and their polar heads 500facing the surrounding aqueous environment (not shown). Unesterifiedcholesterol 600 is found largely within the phospholipid monolayer.Apolipoprotein 700 is disposed within phospholipid molecules 300.Artificial triglyceride emulsion particles have essentially identicalstructures, either with or without protein.

[0039] Liposome 200 is comprised of phospholipid molecules 300 forming aphospholipid bilayer, e.g. one larnella, either with or without protein,in which fatty acyl side chains 400 face each other, the polar headgroups 500 of the outer leaflet face outward to the surrounding aqueousenvironment (not shown), and the polar head groups 500 of the innerleaflet face inward to the aqueous core 202 of the particle 200.Depending on the composition of particle 200, phospholipid bilayers canhave a large capacity for unesterified cholesterol and otherexchangeable material and components thereof. As illustrated in FIG. 1there is no sterol. Typically, such liposomes can pick up unesterifiedcholesterol from other lipid bilayers, such as cell membranes, and fromlipoproteins. Liposomes also pick up proteins and donate phospholipidsand other exchangeable material and components thereof. Liposomes canalso have multilamellar structures, in which the bilayers are containedwithin the environment encapsulated by an outer bilayer to form multiplelamellae. The multiple lamellae can be arranged concentrically, like thelayers of an onion, or in another variant non-concentrically.

[0040]FIGS. 3 and 4 illustrate plasma LDL cholesteryl esterconcentrations in response to injections of LUVs, SUVs or saline overtime. Rabbits were intravenously injected on days 1, 3 and 5 asindicated by arrows 302, 304, and 306 respectively, with a bolus of 300mg of phosphatidylcholine per kg of body weight or a matched volume ofsaline. The phosphatidylcholine was pharmaceutical grade egg PC, in theform of either large unilamellar vesicles (LUVs) having diameters ofapproximately 100 NM (preferably ≅120 NM) prepared by extrusion (LUVswere measured at about 120 NM (123±35 NM and the extrusion membrane hadpores of about 100 NM in diameter) or small unilamellar vesicles withdiameters of approximately 30 NM (preferably 35 NM) prepared bysonication. (SUVs were measured in the range of 34±30 NM.) Blood wasdrawn just before each injection and on the sixth day at sacrifice.Plasma LDL cholesteryl ester concentrations were determined by a gelfiltration assay of the plasma with an in-line enzymatic assay forcholesteryl ester. Means ±SEMs are shown in FIG. 3. Animals infused withSUVs showed significantly higher plasma concentrations of LDLcholesteryl ester at days 3, 5, and 6 compared to either LUV-infused orsaline infused animals. FIGS. 2-8, 10-15, 24 and 28 illustrate data fromthe same experiment in which injections were made on days 1, 3, and 5and then livers were taken. Gel filtration was done of plasma to measurelipid contents of individual lipoprotein classes. FIG. 2 illustrates atable of hepatic mRNA content (pg/Ig) for CETP, HMG-CoA R (hydroxymethylglutaryl coenzyme A reductase), LDL receptors, and cholesterol 7alpha-hydroxylase; and LDL ChE (low density lipoprotein cholesterylester) for the rabbits given saline (HEPES buffered saline) (rabbits1-4), LUVs (rabbits 5-8), and SUVs (rabbits 10-12) for the experimentdescribed for FIGS. 3 and 4. Rabbit 13 is the “Mix” rabbit.

[0041]FIG. 4 shows an animal labeled as mix. “Mix” refers to a singleanimal that received SUVs on day 1, 3 and 5, but also one injection ofLUVs on day 3. Before this injection of LUVs, the plasma concentrationof LDL cholesteryl ester rose, but after the injection of LUVs, the LDLconcentration fell, despite continued injections of SUVs.

[0042]FIG. 5 illustrates LDL receptor mRNA levels in liver in responseto injections of LUVs, SUVs or saline over time. The rabbits describedabove were sacrificed at day 6, and samples of liver were snap-frozen inliquid nitrogen. mRNA was extracted, and rabbit mRNA for the LDLreceptor was quantified by an internal standard/RNase protection assay(Rea T. J. et al. J. Lipid Research 34:1901-1910, 1993 and Pape M. E.,Genet. Anal. 8:206-312, 1991). Means ±SEMs are shown in FIG. 5. Animalsinfused with SUVs showed significant suppression of hepatic LDL receptormRNA compared to LUV-infused or saline-infused animals. Suppression ofhepatic LDL receptor mRNA reflects parenchymal cell overload withsterol, and is a potentially harmful alteration from normal hepaticcholesterol homeostasis. In contrast, LUV-infused animals showed thehighest levels of hepatic LDL receptor mRNA, though the increase abovethat seen in the saline-infused animals did not reach statisticalsignificance. The liver from the “Mix” animal described above showed avalue of 5.28 pg LDL receptor mRNA/microgram which is closer to theaverage value in the saline group than in the SUV group. Thus, LDLreceptor mRNA was stimulated by the single injection of LUVs despiterepeated injections of SUVs.

[0043]FIG. 6 illustrates HMG-CoA reductase mRNA levels in liver inresponse to injections of LUVs, SUVs, or saline. The experimentaldetails are those as referenced above. Animals infused with SUVs showedsignificant suppression of hepatic HMG-CoA reductase mRNA compared toLUV-infused or saline infused animals. Suppression of hepatic HMG-CoAreductase mRNA reflects parenchymal cell overload with sterol, which canbe a potentially harmful alteration from normal hepatic cholesterolhomeostasis. In contrast, LUV-infused animals showed the highest levelsof hepatic HMG-CoA reductase mRNA, though the increase above that seenin the saline-infused animals did not reach statistical significance.

[0044] The “mix” animal showed a value of 0.50 pg HMG-CoA reductasemRNA/microgram, which is essentially identical to the average value inthe saline group (0.51) and substantially higher than the value in theSUV group (0.27). Thus, HMG-CoA reductase mRNA was stimulated to itsnormal value by the single injection of LUVs, despite repeatedinjections of SUVs.

[0045]FIG. 7 illustrates cholesteryl ester transfer protein mRNA levelsin liver in response to injection of LUVs, SUVs, or saline. Theexperimental details are those as referenced above. Animals infused withLUVs showed significant suppression of hepatic CETP mRNA compared to SUVinfused or saline infused animals. Suppression of CETP mRNA producechanges in the plasma lipoprotein profile usually associated withreduced risk of atherosclerosis. The “mix” animal showed a value of 3.18pg CETP mRNA/microgram, which is closer to the average value in the LUVgroup than in the SUV or saline groups. Thus, CETP mRNA was suppressedby the single injection of LUV's despite repeated injections of SUVs.

[0046]FIG. 8 illustrates cholesterol 7-alpha hydroxylase mRNA levels inliver in response to injections of LUVs, SUVs, or saline. Theexperimental details are those as reference above. Animals infused withSUVs showed suppression of hepatic 7-alpha hydroxylase mRNA compared toLUV infused or saline infused animals. Suppression of 7-alphahydroxylase can be a potentially harmful alteration from normal hepatichomeostasis. In contrast, LUV-infused animals showed the highest levelsof hepatic 7-alpha hydroxylase mRNA, though the increase above that seenin the saline infused animals did not reach statistical significance.The “mix” animal showed a value of 0.51 pg 7-alpha hydroxylasemRNA/microgram, which is higher than the average value in the SUV group.Thus, 7-alpha-hydroxylase mRNA was stimulated by the single injection ofLUVs, despite repeated injections of SUVs.

[0047]FIG. 10 illustrates unesterified cholesterol concentrations inwhole plasma in response to injections of LUVs, SUVs, or saline overtime. The experimental details are those as referenced above. Asindicated by this figure, LUVs and SUVs significantly raised the plasmaconcentrations of unesterfied cholesterol indicating mobilization oftissue stores. The LUVs raised the concentration of unesterifedcholesterol more than did the SUVs.

[0048]FIG. 11 illustrates esterified cholesterol concentrations in wholeplasma in response to injections of LUVs, SUVs or saline over time. Theexperimental details are those as referenced above. SUVs raised theplasma concentrations of cholesteryl ester on days 3, 5, and 6. FIG. 12duplicates the information contained in FIG. 3.

[0049]FIG. 13 illustrates plasma VLDL esterified cholesterolconcentrations in response to injections of LUVs, SUVs, or saline. SUVsincreased the plasma concentration of VLDL cholesteryl ester over thatseen in the saline of LUV treated groups. The “mix” animal showed aplasma VLDL cholesteryl ester concentration at day 6 of 2.4 mg/dl, whichis lower than the average value in the SUV group. The experimentaldetails are those as referenced above.

[0050]FIGS. 14 and 15 illustrate HDL esterified cholesterolconcentrations in response to injections of LUVs, SUVs, or saline. Theexperimental details are those as referenced above as in FIG. 2.Suitable phospholipid can be obtained from Avanti Polar Lipids, NipponOil and Fat in Japan and Princeton Lipids, as well as other suppliers.LUVs are made through an extruder that is commercially available. SUvscaused a small but statistically significant rise in HDL cholesterylester concentrations on days five and six.

[0051]FIG. 16 illustrates the time course of cholesterol mobilizationfollowing an LUV injection into control or apoe KO (knock-out) micecommercially available from Jackson Laboratories, in Bar Harbor, Me.Control (C57/BL6) and apolipoprotein E knock-out mice were injected attime zero with a single bolus of 300 mg LUV phospholipid/kg body weight.The LUVs contained a tracer amount of labeled cholesterylhexadecylether, which remains on the liposomes after injection into amouse. Displayed data are for concentrations of total cholesterol, i.e.esterified plus unesterifed, in whole plasma. The rise in both sets ofanimals indicated that LUVs mobilize cholesterol into the plasma, evenin the presence of a severe genetic hyperlipidemia.

[0052]FIG. 17 illustrates the time course of LUV clearance in controlmice and apoe mice. The experimental details are as described in FIG.16. The clearance of LUVs from the plasma is unimpaired in the apoeknock-out mice, indicating mobilization (FIG. 16) and disposal (FIG. 17)of cholesterol even in the presence of a severe genetic hyperlipidemia.This indicates the usefulness of this preparation in hyperlipidemias.

[0053]FIG. 18 illustrates exemplary applications for the compositionsand methods of the present invention in humans. The therapeutic targetsof the compositions and methods presented herein are lipid-rich, ruptureprone plaques, critical stenosis, post-angioplasty re-stenosis,atherosclerosis in general, and any membrane, cell, tissue, organ, andextracellular region and/or structure, in which compositional and/orfunctional modifications would be advantageous.

[0054]FIG. 19 illustrates a perspective view of an improved hemodialysissystem of the present invention and improved method of hemodialysis.Blood is taken from a site for circulatory access (shown here as arm1900) and transported into a cell-plasma separator 1910. The plasma isthen transported to a dialysis chamber 1920 and is divided into at leasttwo compartments that are separated by a semi-permeable membrane 1930.One side of the membrane 1930 is the patient's plasma 1940 and on theother side is the dialysate 1950. Selected molecules exchange across themembrane 1930 depending on the characteristics of the membrane (charge,pore size, etc.). The device 1960 comprises a device for adding lipidacceptors to the dialysate and for sampling the dialysate to allowassays of cholesterol, phospholipid, and other components, such asacceptors, specific lipoproteins, specific components, and to monitortreatment. Extraction of plasma cholesterol or other extractablematerial comprises several possibilities: 1) acceptors are disposed inthe dialysate that do not cross membrane 1930 into plasma; 2) theacceptors do cross membrane 1930 and are either left in the plasma andreturned to the patient or are separated from plasma before it isreturned to the patient; and/or 3) immobilized acceptors on a sheet(such as membrane 1930 itself), on beads, and/or on the walls of thechamber 1920. Plasma thus treated is returned to the patient, usuallyafter having being re-mixed with the blood cells. As noted, cholesterolacceptors can be added at any stage, as an example, a device 1970comprises acceptors and for adding acceptors to plasma shortly beforeits return into the patient is also illustrated in FIG. 19. It isfurther understood that contaminating cellular material, such asplatelets, in the plasma will also become cholesterol depleted inendogenous lipids and enriched in phospholipid. It is further understoodthat all acceptors mentioned throughout this application may acceptmolecules in addition to cholesterol and may donate material as well.

[0055] The cellular concentrate from the cell-plasma separator 1910 canthen be treated in any of several ways before being returned to thepatient: 1) returned to the patient with no further treatment (thisincludes being mixed with plasma that has been treated as above); 2)transferred to a second dialysis chamber (not shown) in which thedialysate contains cholesterol acceptors to lipid deplete the cells ofendogenous lipids, such as cholesterol, before their return to thepatient; 3) mixed with a suspension or solution of lipid acceptors tolipid deplete the cells of endogenous lipids, then either returned tothe patient with the acceptors or option 1) and option 2) above can beperformed with all cell types together, or after further separation intospecific cell types (for example, purified platelets could be lipiddepleted of endogenous lipids, such as cholesterol, and enriched inliposomal lipids). Options 2) and 3) can be performed with periodicassays of cellular cholesterol, phospholipid, fluidity, viscosity,fragility, cell composition and/or cell function. Devices 1960, 1970include an apparatus that allows for the periodic sampling of cellsduring treatment. As with plasma, lipid acceptors can be added at anystage of the treatment. All fluids, e.g. plasma and concentrated cells,are moved by gravity, mechanically, by manual manipulation (a syringe),or with pumps as needed. Of course, it is understood that blood can bedrawn for processing from any appropriate part of the body.

[0056]FIG. 20 illustrates a perspective view of an improved peritonealdialysis system 2000 and method of peritoneal dialysis. Patient'sabdomen 2010 (FIGS. 20-21) receives peritoneal dialysate 2020 stored incontainer 2030 into the peritoneal cavity through incision 2040 by wayof channel 2050. Lipid acceptors and/or cholesterol acceptors 2060 areoptionally disposed in container 2070. In another variant, lipidacceptors are added to dialysate 2020; added to container 2030 inconcentrated form shortly before infusion; added as shown to the streamof fluid entering the peritoneal cavity; or infused by a separate portalof entry into the patient by any effective route. Throughout thisapplication, it is understood that all acceptors may accept molecules inaddition to cholesterol and may donate material such as phospholipidsand antioxidants.

[0057]FIG. 21 illustrates a perspective view of a variant of an improvedperitoneal dialysis system with assaying means 2100 and method ofperitoneal dialysis and analysis of spent fluid. Container 2110 acceptsspent fluid from abdomen 2010 by way of channel 2120. The device 2110provides access to diagnostic samples of spent dialysate to allow forassay of cholesterol, phospholipid, and other parameters as describedherein showing the efficacy of the treatments described. Optionally,assay syringe 2130 is inserted by way of access portal 2140 into channelor tube 2120, or into container 2110, and optional pumps (not shown) areused to move the various fluids to appropriate locations for assaythereof.

[0058]FIG. 22 illustrates a perspective view of an improved cardiaccatheterization and/or angioplasty system 2200 and method of cardiaccatheterization and/or angioplasty. Patient 2210 undergoes cardiaccatherization and/or angioplasty. The patient intravenously receiveseffective doses of lipid acceptors or cholesterol acceptors 2230co-administered with said treatment(s) from container 2220.Intraarterial access of a catheter for coronary angiography and/orangioplasty allows for ready co-administration of cholesterol acceptorsand administration of diagnostic agents such as cholinergic agents, toassess vascular function.

[0059]FIG. 23 illustrates a perspective view of a variant of an improvedcardiac catheterization and/or angioplasty system 2300 and method ofcardiac catheterization and/or angioplasty. Catherization and/orangioplasty catheter 2310 has apertures 2320 that allow for the egressof cholesterol acceptors therefrom. In a variant, catheter 2310 has apermeable membrane that allow for the egress for cholesterol acceptorstherefrom. Phantom arrows 2330 indicate egress sites for cholesterolacceptors and/or diagnostic agents. Sites 2340 indicate entry sites forthe acceptors or agents. The balloon on the device 2300 can be replacedor supplemented with other devices or can form an inner balloon layerdisposed within an outer balloon layer. The acceptors are disposedbetween the inner and outer flexible balloon layers. Upon expansion ofsaid inner balloon layer a force is exerted against the fluid orgel-like acceptors forcing the acceptors out of the sites 2320, and intodirect contact (forcefully) against arterial lesions more locallydirecting the treatment. It will be appreciated that this variant of theinvention provides for maximal penetration of the acceptors into thearterial lesions. The infusions can be accomplished by gravity, manualmanipulation of a syringe, or by mechanical infusion pump 2350. The samemethod and system can be utilized with standard vascular imagingtechniques or vessels that include the femorals, carotids, andmesenteric vessels by way of example.

[0060] Patient 2210 undergoes cardiac catherization and/or angioplasty.The patient intravenously receives effective doses of cholesterol orlipid acceptors 2230 co-administered with said treatments(s) fromcontainer 2220. Intraarterial access of a catheter for coronaryangiography and/or angioplasty allows for ready co-administration oflipid or cholesterol acceptors and administration of diagnostic agentssuch as cholinergic agents, to assess vascular function.

[0061] Container 2110 accepts spent fluid from abdomen 2010 by way ofchannel 2120. The device 2110 provides access to diagnostic samples ofspent dialysate to allow for assay of cholesterol, phospholipid, andother parameters as described herein showing the efficacy of thetreatments described. Optionally, assay syringe 2130 is inserted by wayof access portal 2140 into channel or tube 2120, and optional pumps (notshown) are used to move the various fluids to appropriate locations forassay thereof.

[0062]FIG. 24 illustrates a graph of hepatic lipid contents in responseto injections of LUVs, SUVs, or saline. The experimental details are asoutlined above. Liver samples were assayed for contents of severallipids: cholesterol ester (CE); triglyceride (TG); unesterifiedcholesterol (Chol); phosphatidylethanolamine (PE); andphosphatidylcholine (PC), which are displayed in units of μg(micrograms) lipid/mg. Lower values of PE and PC in the SUV-treatedanimals were produced; thus, the Chol:phospholipid ratios in theseanimals was higher than in the other groups.

[0063]FIG. 25 illustrates cholesterol ester concentrations followingrepeated injections of SUVs or LUVs (30 mg/kg) in NZW rabbits (NewZealand White rabbits). The arrows indicate times of phospholipidinjection here on days 0, 3 and 5. For a given phospholipid dose, LUVspromote a greater rise in plasma free cholesterol concentrations.

[0064]FIG. 26 illustrates plasma free cholesterol concentrationsfollowing repeated injections of SUV or LUV (300 mg/kg) in NZW rabbitsin the same experiment as in FIG. 25, arrows indicate times ofphospholipid injection. Repeated injections of LUV, unlike SUV, do notprovoke a dramatic rise in CE concentrations in plasma.

[0065] The rise in plasma CE concentrations that results from thedelivery of excess cholesterol to the liver may be the consequence oftwo processes. It may involve an overproduction of CE-rich particles oran impaired clearance of CE-rich lipoproteins. Overproduction of CE-richparticles that occurs following SUV infusions may result in the plasmaor in the liver. In plasma, LCAT acts on small unilamellar phospholipidvesicles or on phospholipid enriched HDL generating CE which may besubsequently transferred by CETP onto LDL. The results with gelfiltration of plasma from animals treated with SUVs indicate that CE iscarried mostly or substantially on LDL. Also, in plasma, removal of apoEfrom VLDL by SUVs will slow the clearance of VLDL, thereby favoring amore efficient conversion into LDL. In the liver, the increased deliveryof cholesterol to hepatocytes during cholesterol mobilization stimulatesan over secretion of apoB, CE-rich lipoproteins.

[0066] In a variant, the rise in plasma CE concentrations observed isthe result of an impaired clearance of CE rich atherogenic lipoproteins.Intravenously administered liposomes that acquire apoe compete with LDLfor LDL-receptor mediated uptake. The delivery of excess cholesterol tothe liver down regulates LDL receptors. The processes responsible for anincrease in plasma CE concentrations are different between the twoliposome preparations. LUVs, unlike SUVs, do not provoke a rise inplasma CE concentrations. LUVs are superior preparations for mobilizingtissue cholesterol without harmful side effects.

[0067] The method and composition of the present invention also providesenrichment of HDL cholesterol esters by SUVs. One contributing processis the stimulation of lecithin cholesterol acyl transferase (LCAT) andother processes related thereto. The ability of SUVs to increase HDLcholesterol ester is the result of stimulation of LCAT and otherprocesses related thereto. LCAT need phospholipid and cholesterol togenerate cholesteryl ester and lysophosphatidylcholine; liposomes cansupply extra phospholipid. The present invention also provides foralterations in lipoprotein (LDL, HDL, etc.) composition and function byLUVs and/or SUVs and/or other acceptors.

[0068] The liposome compositions described herein and methods utilizingsame also include the liposomes picking up endogenous apoE and henceblocking cellular uptake of LDL. The liposomes pick up apolipoproteins,such as apoE and apoA-I, and that this alters or enhances theirfunctions. For example, the uptake of endogenous apoA-I enhances theability of liposomal derived phospholipid to pick up cholesterol, andthe uptake of endogenous apoE would allow the liposomes to block certainpathways for arterial uptake of lipoproteins. All of this is in thecontext of controlling LDL levels and hepatic gene expression andcholesterol homeostasis.

[0069] LUVs and SUVs deliver cholesterol to different regulatory poolswithin the liver. This conclusion is supported by the differences inhepatic gene responses and CETP mRNA is suppressed: the LDL receptormRNA is unaffected or increased by LUVs but suppressed by SUVs; and CETPis suppressed by LUVs, but unaffected by SUVs. Further, it is understoodthat the arterial lesions referenced herein include, by way of example,critical stenoses.

[0070] The key points about LUVs and atherosclerosis are illustrated inFIG. 9. The practical benefits of using LUVs as a treatment foratherosclerosis are that they are straight forward to manufacture, andnon-toxic even at very high doses. Mechanistically, LUVs promote reversecholesterol transport in vivo without provoking a rise in LDLconcentration, and LUVs are an optimal preparation.

[0071] The compositions that are used herein can direct clearance awayfrom hepatic parenchymal cells. And the various methods described hereinare utilized with slow infusions of the compositions described, so thathepatic cells are not cholesterol overloaded even if clearance byparenchymal cells occurs. Further, HDL is also controlled by CETP genesuppression.

[0072] As described herein assays are performed by: assaying fastingplasma triglyceride to estimate VLDL concentrations; assaying plasmacholesterol (free and ester, or total minus free=ester); precipitatingLDL (& VLDL) with polyanions-cations; assaying the supernatant which isHDL; and computing LDL's (whole plasma value minus VLDL−HDL) sterol (orsterol ester) in whole plasma. Liposomes will precipitate withpolyanions-cations; or optionally assaying the ester which liposomesmostly lack. Other assays include electrophoresis, chromatography,immune assays, electron microscopic assays, functional assays,structural assays, and compositional assays.

[0073] In the dialysate of the present invention, any liposomes oremulsions could be used as long as it's a cholesterol acceptor andeither it does not raise LDL or it is not returned to the patient'scirculation. In either case, one would need to assay plasma LDL and theplasma concentration of the acceptors, and plasma concentrations ofother atherogenic lipoproteins.

[0074] With respect to the methods that require delivering thecholesterol to the liver at a slow rate, or in low doses administrationmight permit small acceptors, such as SUVs, to be used without LUVsprovided LDL levels as levels of other atherogenic lipoproteins aremonitored and regulated. To avoid disrupting hepatic cholesterolhomeostasis, the entrapped drug as described herein need not be given atlow doses, but rather the encapsulating liposome or emulsion is given inlow doses; the drug could be present at high amounts within a smallnumber of liposomes or a small mass of liposomal lipid.

[0075] Alterations in HDL size, composition and function can beaccomplished by administering high or even truly low doses of largeand/or small liposomes that have little or no sterol. Liposomes withoutsterol, when given in low doses are easily broken apart by HDL and HDLapolipoproteins and then pieces are incorporated into the HDL fractionof plasma enriching it in phospholipid. Such small doses, e.g. 10-100mg/kg/dose, even of SUVs without LUVs or drugs to lower LDL levels, areunlikely to raise plasma LDL levels, although periodic monitoring wouldbe prudent.

[0076] Also, the method as disclosed herein of altering LDL compositionwithout increasing LDL concentration would be to enrich the compositionwith phospholipids, like POPC (palmitoyloleylphosphatidylcholine), thatare resistant to oxidation, enrich the composition with anti-oxidants,deplete unesterified cholesterol, and reduce cellular or arterial uptakeof oxidized LDL by phospholipid enrichment.

[0077] Liposomes up to about 1000 NM or so would work in the presentinvention. Larger liposomes would also work but extraction of tissuelipoprotein may be less efficient. It is further possible to concentrateor dry compositions of the present invention. These preparations arethen diluted or reconstituted at the time of therapy or administration.In this variant, a two component kit comprising the active material anda dilutent is provided. Inclusion of phosphatidyl glycerol (PG) to makethe liposomes negatively charged, or charge other components of thecomposition, to prevent aggregation during storage is also provided.

[0078]FIG. 27 illustrates alterations in plasma components afterrepeated injections of SUVs. Watanabe Heritable Hyperlipidemic (WHHL)rabbits were given intravenously 1000 mg of SUV phospholipid per kg ofbody weight, or the equivalent volume of saline, on Monday, Wednesday, &Friday of each week for three weeks (nine doses total). Three days afterthe final dose, blood samples were taken, and plasma components werefractionated by size by passage over a Superose-6 gel-filtration column.Eluents were read by an in-line spectrophotometer. The tracing on theright is from a saline-injected rabbit, and shows VLDL around fractions#17-18, and LDL around fraction #27. The tracing on the left is from anSUV-injected rabbit, and shows VLDL with persistent liposomes aroundfraction #16, and LDL-sized particles around fraction #25. The tracingsindicate an increase in the amount of LDL-sized particles after repeatedinjections of SUVs consistent with an increase in LDL, which is aharmful effect. Because WHHL rabbits have a genetic lack of LDLreceptors, this result indicates that SUVs disrupt hepatic cholesterolhomeostasis not just by suppressing LDL receptors (FIG. 5), but also bymechanisms independent of LDL receptors (FIG. 27). LUVs avoid both LDLreceptor-dependent and independent disruptions.

[0079]FIG. 28 illustrates an agarose gel electrophoresis of whole plasmafollowing repeated injections of LUVs, SUVs, or saline. Experimentaldetails are referenced in FIGS. 2-8 & elsewhere herein. Four-μL plasmasamples from two rabbits in each group at day 6 were electrophoresedthrough 1% agarose then stained for lipids with Sudan black. O: origin.β: migration of an LDL standard. The SUV-mediated increase in LDLconcentration is illustrated by the darker but otherwise unremarkableβ-band in those lanes. SUVs in plasma exhibited a mobility ahead of LDL,owing to their acquisition of plasma proteins, chiefly from HDL. Incontrast, plasma LUVs exhibited essentially the same mobility as freshlyprepared, protein-free vesicles, i.e., just above the origin (O),indicating a substantial absence or reduction of acquired proteins onthe LUVs.

[0080] Based on the electrophoretic mobilities in FIG. 28,quantification of the acquisition of protein by LUVs versus SUVs wasobtained. LUVs and SUVs were incubated with human HDL in vitro for 4hours at 37° C, then separated from the HDL by gel filtrationchromatography and assayed for protein and phospholipid. LUVs acquired1.09 μg of protein per mg of liposomal phospholipid, whereas SUVsacquired 40.4 μg/mg, i.e., almost 40 times as much. Thus, the two typesof liposomes exhibit a striking quantitative difference in proteinadsorption. SUVs, but not LUVs, avidly strip apoe from VLDL, therebyslowing its clearance from plasma and favoring its conversion to LDL. Inaddition, adsorbed proteins play a role in directing the SUVs into ahepatic metabolic pool that disrupts hepatic cholesterol homeostasis,whereas LUVs are not directed into such a pool. Liposomes, emulsions, orany other particles or compounds that extract tissue lipids but do notacquire large amounts of plasma proteins behave similarly to LUVs inthese regards.

[0081] Specific vascular genes affected by cholesterol loading of cellsinclude genes for prolyl-4-hydroxylase; hnRNP-K; osteopontin (there maybe a role for oxidized lipids in provoking arterial calcifications); andMac-2. The methods of regulating these genes described herein effectrestoration of normal vascular or arterial function. Elevated expressionof prolyl-4-hydroxylase (an enzyme in the synthesis of collagen, acomponent of fibrotic plaques) and hnRNP-K (identified in pre-mRNAmetabolism and cell cycle progression) messages were found in aorticsmooth muscle cells after cholesterol feeding. These would normalizeafter the liposome treatments described herein. Other genes or enzymesthat are abnormal with cholesterol-loading and should normalize withliposome treatment as described herein include osteopontin, nitric oxidesynthase (NOS), adhesion molecules, chemoatractants, tissue factor,PAI-1 (plasmidigen activator inhibitor), tPA (tissue plasmidigenactivator) and Mac-2 (Ramaley et al. 1995). Other genes affected bycholesterol, cholesterol loading, oxidized lipids would also becorrected.

[0082] Many examples of small acceptors such as SUVs,apolipoprotein-phospholipid disks, and HDL are commercially availableand can be used in the invention. Kilsdonk EP et al. Cellularcholesterol efflux mediated by cyclodextrins, J. Biol. Chem.270:17250-17256, 1995. By way of further example, another small acceptorincludes the cyclodextrins. Small acceptors (specifically HDL) shuttlecholesterol from cells to liposomes. Cyclodextrins and also other smallacceptors can shuttle cholesterol and other exchangeable material fromcultured cells to LUVs, which substantially increases the removal anddonation of material between cells and LUVs.

[0083] Examples of anti-hyperlipidemic drugs include fibric acidderivatives, HmG CoA reductase inhibitors, Niacin, probucol, bile acidbinders, other drugs and combinations thereof. Anti-hyperlipidemictreatments also include LDL, apheresis, leal bypass, livertransplantation and gene therapy.

[0084] The data presented in this application support three possibleexplanations for the difference in metabolic response to LUVs versusSUVs. The three mechanisms act separately or in combination. First, LUVsare taken up largely by Kupffer cells, whereas SUVs are primarilydirected towards hepatic parenchymal cells. This is partly a mechanicalconsequence of hepatic architecture: hepatic endothelial fenestrae areoval openings of about 100×115 nm, through which SUVs of 30-nm diameteror so can readily pass and gain access to parenchymal cells. Largeparticles, such as large liposomes, of sufficient diameter will not passeasily, and are cleared instead by the macrophage Kupffer cells thatline the liver sinusoids. While SUVs also have access to Kupffer cells,their sheer number (˜10 times as many SUVs as LUVs per mg ofphospholipid) appears to saturate the reticuloendothelial system, and soparenchymal cells predominate in their clearance. Other methods todirect artificial particles away from parenchymal cells are alsoavailable, such as by changing the particle structure or composition,including charge and specific ligands for cell-specific binding.

[0085] Cholesterol clearance pathways mediated by parenchymal versusKupffer cells have distinct metabolic consequences. Direct delivery ofcholesterol to parenchymal cells by SUVs suppresses sterol-responsivemessages (FIGS. 5, 6, & 8). Delivery of cholesterol to Kupffer cells canbe followed by gradual transfer of lipid to parenchymal cells, forexample, via the extensions of Kupffer cells that reach down through thespace of Disse to make physical contact with parenchymal cells. The rateof sterol delivery to the parenchymal cells by transfer from Kupffercells can be slower than by direct uptake; the chemical form of thesterol may be altered by the Kupffer cells before transfer; there isother cell-cell communication; and, based on other pathways for lipidtransfer amongst liver cells, the process of transfer from Kupffer toparenchymal cells may be regulated, whereas SUV clearance does notappear to be.

[0086] The second contributing explanation for the difference inmetabolic response to LUVs versus SUVs is based solely on differences inthe kinetics of their delivery of cholesterol to the liver. LUVs arecleared from plasma somewhat more slowly than are SUVs, and therebyproduce a relatively constant delivery of cholesterol mass to the liverfrom the time of injection until the bulk of injected material iscleared. SUVs are cleared more rapidly, thereby delivering a large bolusof cholesterol mass to the liver several hours after each injection,which is followed by the sustained rise in plasma concentrations ofcholesteryl ester and atherogenic lipoproteins. The slow, steadydelivery by LUVs avoids disrupting hepatic cholesterol homeostasis,while the more rapid uptake of SUV cholesterol overwhelms the ability ofthe liver to maintain homeostasis, thereby provoking suppression ofhepatic LDL receptors. Other methods to deliver artificial particles ortheir components to the liver at a proper rate are also available, suchas by changing the particle structure or composition, including chargeand specific ligand for cell-specific binding.

[0087] The third contributing explanation is based on the strikingquantitative difference in protein adsorption between the two types ofvesicles (FIG. 28), which, in that particular experiment, was a resultof their distinct surface curvatures. Thus, SUVs, but not LUVs, wouldavidly strip apoE from VLDL, thereby showing its clearance from plasmaand favoring its conversion to LDL. SUVs that acquire apoE will competewith VLDL, LDL, and other particles for receptor mediated uptake by theliver. Also, adsorbed apoproteins can play a role in directingphospholipid vesicles to different hepatic metabolic pools. Othermethods to reduce protein uptake by artificial particles are alsoavailable, such as by changing the particle structure or composition,including charge and specific ligands for cell-specific binding.

[0088] Overall, given the observation that cholesteryl ester and LDLconcentrations do not increase after delivery of large amounts ofcholesterol and other exchangeable material to the liver by LUVs, it wasapparent that delivery was to a specific metabolic pool or pools withunique properties that do not increase plasma concentrations ofatherogenic lipoproteins or harmfully disturb hepatic cholesterolhomeostasis, including the regulation of genes and other functions.Thus, these inventions can be regarded in part as a unique deliverysystem that brings original particle components, such as phospholipid,plus material acquired by the particles, such as cholesterol, to aspecific delivery site for harmless disposal and other additionalbenefits. The delivery system with these characteristics will be usefulin any situation whatsoever in which control of hepatic cholesterolhomeostasis, hepatic phospholipid homeostasis, and hepatic metabolism ingeneral is advantageous.

[0089] For example, in a situation in which it is desirable to modifyerthyrocyte lipids, a straightforward approach would be to administerartificial particles that can donate and remove the appropriate lipids.If SUVs are used for this purpose, however, they will transportcholesterol and other material to the liver in a harmful manner, to thewrong pool and/or at the wrong rate, and this will cause increases inplasma concentrations of atherogenic lipoproteins, which is anundesirable side-effect that would preclude this approach. In contrast,the use of large liposomes or other particles with similar propertieswill result in the proper delivery of original and acquired material, tothe proper pool(s) at a proper rate, so that the desired effect(modification of erythrocyte lipids) can be achieved without harmfulincreases in plasma concentrations of atherogenic lipoproteins.

[0090] As another example, it can be desirable to modify infectiousagents, such as bacteria, fungi, and viruses, using the compositions andmethod described herein. Administration of large liposomes or otherparticles with similar properties will remove and donate exchangeablematerials to and from these infectious agents, and then the administeredparticles will be delivered to the proper pool(s), so that the desiredeffect can be achieved without harmful increases in plasmaconcentrations of atherogenic lipoproteins.

[0091] As another example, a valuable therapy may provoke an increase inplasma concentrations of atherogenic lipoproteins as an unwantedside-effect. Administration of large liposomes or other particles withsimilar properties will alter this response through the delivery oflipids and other material to the proper hepatic metabolic pool. The datawith the “Mix” animal provides a specific example of this effect (FIG.4).

[0092] There are several mechanisms for affecting arterial uptake,accumulation, and retention of lipoproteins. Liposomes can pick up apoEfrom atherogenic lipoproteins, thereby reducing lipoprotein binding toarterial cells and also competing for binding to arterial cells.Finally, alterations in LDL size and/or composition affect its bindingto extracellular matrix and affect subsequent, harmful alterationswithin the arterial wall, for example, susceptibility to oxidation orenzymatic modifications.

[0093] The action or mode of operation of large acceptors, such as largeliposomes, can be aided by small acceptors, and vice-versa, and thisapplies to both endogenous (e.g., HDL) and exogenous (e.g.,apoprotein-phospholipid complexes) small acceptors. Large acceptorspenetrate poorly into the interstitial space and appear to inefficientlyapproach the cell surface under certain circumstances. These effectsimpede their uptake and donation of exchangeable material frommembranes, cells, tissues, organs, and extracellular regions andstructures. Small acceptors do penetrate well into the interstitialspace and are able to approach the cell surface, thereby allowingefficient uptake of exchangeable material. Small acceptors have majordisadvantages, however. They have a very limited capacity to acquire ordonate material (even though the initial rate of acquisition or donationis rapid, until their capacity becomes saturated) and, once they haveacquired material, they deliver it to the liver in a way that disruptshepatic cholesterol homeostasis.

[0094] Large acceptors and small acceptors together, however,synergistically overcome each other's drawbacks through at least threemechanisms. First, the large acceptors act as a sink (or supply) forexchangeable material, while the small acceptors act as a shuttle thatsiphons material from peripheral stores to the large acceptors and inthe other direction. Thus, for example, the small acceptors penetratetissue, acquire (and/or donate) material from the tissue, and theircapacity becomes at least partly saturated. They leave the tissue andencounter the large acceptors in the plasma, at which point the smallacceptors are stripped of tissue lipids. The capacity of the smallacceptors is thereby restored, so that when they return to the tissue,they can acquire (and/or donate) more material. This cycle can continuemany times. Second, the large acceptors can re-model some smallacceptors. For example, large acceptors can donate phospholipid to HDL,which increases the capacity of HDL acquire tissue cholesterol and othermaterial. Third, as noted elsewhere, the presence of large acceptors canblock or reduce the harmful disruptions in hepatic cholesterolhomeostasis caused by the small acceptors.

[0095] Large liposomes avoid raising plasma concentrations ofatherogenic lipoproteins in general, not just LDL. This list includesall lipoproteins that contain apolipotrein B (apoB), such as LDL, IDL,VLDL, Lp(a), β-VLDL, and remnant lipoproteins.

[0096] Immune cells are also the targets for depletion using the methodsand modes of operation disclosed herein. It is understood thatadministration of an HMG-CoA reductase inhibitor, pravastatin, tocardiac transplant recipients reduced their natural-killer-cellcytotoxicity in vitro, reduced episodes of rejection accompanied byhemodynamic compromise, reduced coronary vasculopathy, reduced plasmaLDL levels (and increased HDL levels), and significantly enhancedone-year survival. The effect on survival was dramatic: in the controlgroup, 22% died in the first year, whereas only 6% died in thepravastatin-treated group.

[0097] Immunologic effects of HMG-CoA reductase inhibitors have beenreported in vitro. These reported immunologic effects include theregulation of DNA in cycling cells, the inhibition of chemotaxis bymonocytes, the regulation of natural-killer-cell cytotoxicity, and theinhibition of antibody-dependent cellular cytotoxicity. Regulation ofsuch inhibitors results from changes in circulating lipids or othereffects and by utilization of the methods and modes of operationdisclosed herein.

[0098] HMG-CoA reductase catalyzes an early step in cholesterolbiosynthesis and is crucial in the synthesis of molecules besidescholesterol. Adding cholesterol to immune cells treated with HMG-CoAreductase inhibitors does not restore function, although the addition ofmevalonate does. Although this suggests that cholesterol depletion isnot directly responsible for the immune effects, the use of liposomes orother acceptors to remove cholesterol from cells increases endogenousconsumption of mevalonate, as the cells try to make more cholesterol. Toimpede the ability of the immune or other cells to make up theircholesterol loss by picking up more LDL or other lipoproteins, themethods and treatment described herein are also be done in conjunctionwith therapies to lower plasma cholesterol concentrations (includingHMG-CoA reductase inhibitors, fibric acids, niacin, bile acid binders,LDL-pheresis, etc.).

[0099] These processes include enhancement of cholesterol removal andreduction of cholesterol influx. Levels of HDL, the apparent naturalmediator of cholesterol removal from peripheral cells, increased in atreated group of patients, and LDL levels were deceased. Theadministration of HMG-CoA reductase inhibitors in vivo usually causesvery tiny changes in reductase enzyme activity: cells simply make moreenzyme to overcome the presence of the inhibitor. They also make moreLDL receptors (especially in the liver) and so LDL levels fall.

[0100] The invention further provides for additives to PD (peritonealdialysis solutions) that reduce the accelerated atherosclerosis thatoccurs in renal failure.

[0101] Chemotaxis of monocytes is an important early event inatherosclerotic lesion development: monocytes become attracted toabnormal arterial lipid deposits, and to cellular products made inresponse to the presence of these deposits, enter the vessel wall,transform into macrophages, internalize the lipid by phagocytosis and/orendocytosis, and become a major component of the so-called lipid-richfoam cells of human atherosclerotic lesions. Thus, inhibition ofmonocyte chemotaxis is important for atherosclerosis as well and can beaccomplished using the methods disclosed herein. Both cellular andhumoral immunity seem to be affected by reductase inhibition: cardiacrejection accompanied by hemodynarnic compromise has often beenassociated with humoral rejection (i.e., that occurring withoutproducing marked lymphocytic infiltration in endomyocardial-biopsyspecimens).

[0102] Pravastatin may interact with cyclosporine [an importantimmunosuppressive drug], which blocks the synthesis of interleukin-2 instimulated T-lymphocytes. The addition of interleukin-2 restored thenatural-killer-cell cytotoxicity and partly restored theantibody-dependent cytotoxicity that were inhibited inlovastatin-treated in vitro cell cultures. A synergy betweencyclosporine and pravastatin explains increased immunosuppression inrecipients of cardiac transplants, whereas patients without transplantswho receive HMG-CoA reductase inhibitors for hypercholesterolemia do nothave clinical immunosuppression.

[0103] Thus, the use of safe cholesterol acceptors with otherimmunosuppressives, such as cyclosporine &/or glucocorticoids (which canalso suppress IL-2) is also contemplated by this invention.

[0104] It is also appreciated that the invention utilizes derivatives ofvarious compounds described herein.

[0105] Pathological specimens from patients with cardiac transplants whohave severe coronary vasculopathy have been reported to have a highcholesterol content. Therefore, early cholesterol lowering withpravastatin may play a part in decreasing the incorporation ofcholesterol into the coronary arteries of the donor heart. Largeliposomes or other cholesterol acceptors are used to accomplish the sameeffect, quickly and directly, alone or in combination, therewith.

[0106] Immune modulations is important in many conditions, not justcardiac transplantation. Areas in which the above approaches could beused also include transplantations of other organs, autoimmune diseases(in which the body's immune system mistakenly attacks the body's owntissues), some infections (in which the immune reaction becomesharmful), and any other situation in which immune modulation would behelpful.

[0107] With respect to infections, modification of the lipid content andcomposition of foreign objects in the body (such as infectious agents)while maintaining normal hepatic cholesterol homeostasis should also bementioned.

[0108] Oxidized lipids alter tissue function and cause damage, includingdecreased EDRF, and increased adhesion molecules, cell damage, andmacrophage chemotaxis.

[0109] There are interactions between LUVs and small acceptors, such asHDL, apoprotein phospholipid complexes, and cyclodextrins. Liposomesremodel HDL into a better acceptor by donating extra phospholipid, andthe small acceptors act as a shuttle, carrying cholesterol efficientlyfrom cells to liposomes. LUVs do not elevate LDL concentrations and donot suppress hepatic LDL receptor gene expression. The medical utilityfor LUVs includes restoring EDRF secretion by endothelial cells. Highcholesterol levels inhibit endothelial release of EDRF not throughcholesterol, but through an oxidized derivative of cholesterol. BecauseHDL itself restores EDRF release, perhaps through the removal ofcholesterol or of oxidized lipids, then liposomes would be able to dothe same (the HDL ferries cellular oxidized lipids to liposomes, forexample).

[0110] The invention provides a method and mode of operation formodifying cellular lipids, including oxidized lipids, without provokinga rise in LDL concentrations or harmfully disturbing hepatichomeostasis. Thus, the LUVs, presumably acting in concert withendogenous (or exogenous) small acceptors of cholesterol (such as HDL),pull oxidized lipids out of peripheral tissues and deliver them to theliver for disposal. Oxidized lipids have a wide range of harmfulbiological effects, including suppression of EDRF release, induction ofcell adhesion molecules, cellular damage, chemotaxis of macrophages, andso forth.

[0111] Oxidized lipids and their harmful effects include decreaseendothelial C-type ANF; increased endothelial PAI-1 and decreased tPAand decreased endothelial thrombomodulin. Liposomes enhance orparticipate in this effect. These changes impair the body's ability todissolve clots. The methods disclosed herein assist in amelioratingthese harmful effects of oxidized lipids. HDL acts in part bytransporting enzymes that inactivate biologically active oxidizedlipids.

[0112] It is understood that oxidized LDL inhibits endothelial secretionof C-type natrizuretic peptide (CNP). It is the lipid component ofoxidized LDL that mediates this effect. Most importantly, HDL blocks theaction of oxidized LDL, presumably by picking up oxidized lipids (e.g.,oxidized cholesterol). Coincubation with high-density lipoprotein (HDL),which alone had no effect on CNP release, significantly preventedOx-LDL-induced inhibition of CNP secretion by endothelial cells (ECs).Analysis by thin-layer chromatography demonstrated that oxysterols,including 7-ketocholesterol, in Ox-LDL were transferred from Ox-LDL toHDL during coincubation of these two lipoproteins. These resultsindicate that Ox-LDL suppresses CNP secretion from ECs by7-ketocholesterol or other transferable hydrophilic lipids in Ox-LDL,and the suppressive effect of Ox-LDL is reversed by HDL.

[0113] Whatever molecule HDL picks up, the presence of liposomes orother acceptors around as described herein will allow it to do a betterjob, because of remodeling of HDL by liposomes & shuttling of oxidizedlipids by HDL from tissues to liposomes (i.e., the liposomescontinuously strip the HDL). Liposomes with an exogenous small acceptorwill also work.

[0114] It is further understood that transferable lipids in oxidizedlow-density lipoprotein stimulate plasminogen activator inhibitor-1 andinhibit tissue-type plasminogen activator release from endothelialcells. As above, it is the lipids in oxidized LDL, such as oxidizedforms of cholesterol, that produce the effect. It is understood thatoxidized low density lipoprotein reduced thrombomodulin transcription incultured human endothelial cells. It is appreciated that oxidized lipidsplay a role in atherosclerosis, and enzymes on HDL that inactivateoxidized lipids may contribute to a protective effect. It iscontemplated that the methods and compositions disclosed herein willhelp this proposed mechanism as well, for example, by removingend-products of these enzymes, by otherwise altering HDL, and byproviding an additional platform for enzyme transport and action.

[0115] As such the use of large liposomes to remove harmful lipids ingeneral (here, oxidized lipids) from peripheral tissues, either directlyor via HDL, which would extract the lipids first, possibly inactivatethem, then deliver them or their break-down products to liposomes in thecirculation is described. Direct methods to assess oxidation andoxidative damage in vivo include for lipids, assays for 8-epiPGF₂alpha;for DNA, assess 8-oxo-2′ deoxyguanosine; generally assess anti-oxidantenzymes in tissues; and assess anti-oxidants levels, such as vitamin E,vitamin C, urate, and reduced/oxidized glutathione.

[0116] Methods relating to and modes for effecting the reverse lipidtransport, from cells, organs, & tissues, including transport ofextracellular material, and any exchangable material in general aredescribed herein. This covers not just cholesterol, but alsosphingomyelin, oxidized lipids, lysophophatidylcholine, proteins, andalso phospholipid donation. Some effects of oxidized material includeincreased calcification in arterial cells as described above and below.

[0117] Three potential differences between large versus small liposometo explain their different effects on LDL and apoB levels include:fenestral penetration (LUV<<SV); rate of clearance (LUV<SUV, so thatLUVs produce a slow, sustained cholesterol delivery to the liver thatmay be less disruptive); and protein adsorption (LUV<<SUV).

[0118] Unesterfied cholesterol increases tissue factor expression bymacrophages. This is extremely important, because it ismacrophage-derived tissue factor that makes the material released byunstable, rupturing plaques such a powerful stimulus for a clot to formthat then blocks the vessel leading to a heart attack. The methods andmodes of operation and compositions of the invention act upon theexpression of tissue factor.

[0119] Poor absorption of proteins by large liposomes affects LDL levelsand/or atherosclerosis by the following mechanisms: 1) acquisition ofapoE from VLDL by small liposomes impairs the removal of VLDL from thecirculation, thereby allowing it to be more efficiently converted intoatherogenic LDL; ii) absorbed proteins on small liposomes direct theseparticles into the wrong metabolic pool within the liver. Polyacrylamidegel electrophoresis shows that liposomes (actually small liposomes)increase the size of LDL. Liposomes are used to alter LDL size,composition and structure to decrease its atherogenicity.

[0120] Other properties of LDL could be changed by administration ofliposomes. For example, liposomes reduce surface unesterifiedcholesterol; reduce surface sphingomyelin; replace surface phospholipidswith POPC which is poorly oxidized; supplement the LDL with antioxidantsthat were added to the liposomes before administration. These changeswould substantially alter arterial entry, retention, modification andatherogenicity of LDL.

[0121] The side-effects controlled are focused on hepatic cholesterolmetabolism, hepatic expression of genes involved in cholesterolmetabolism, and plasma concentrations of cholesterol-rich atherogeniclipoproteins that contain apolipoprotein B (chiefly, LDL). Reversetransport of sphingomyelin, for example, changes hepatic cholesterolmetabolism (cellular sphingomyelin affects the intracellulardistribution of cholesterol, and hence its regulatory effects; alsosphingomyelin is a precursor to ceramide, which mediates intracellularsignaling), though large liposomes appear to avoid any problems in thearea. The same holds true for reverse transport of oxidized forms ofcholesterol (they are even more potent that unoxidized cholesterol insuppressing LDL receptor gene expression). Cyclodextrins do not pick upphospholipids.

[0122] Liposomes pick up any exchangeable lipid (actually, anyexchangeable amphipathic or hydrophobic material, which includes lipidor protein or anything else with these characteristics). This includessphingomyelin, oxidized or modified lipids, such as oxidized sterols andphospholipids. Typically, such liposomes can pick up unesterifiedcholesterol and other exchangeable material from other lipid bilayers,such as cell membranes, and from lipoproteins. Liposomes also pick upproteins and donate phospholipids. During and after these modifications,the liposomes are removed from the plasma, chiefly by the liver.Throughout this application, we will refer to this general process as“reverse lipid transport”, although it is understood that anyexchangeable material in tissues, blood, or liposomes could participate.Specific examples of exchangeable material include unesterifiedcholesterol, oxidized forms of cholesterol, sphingomyelin, and otherhydrophobic or amphipathic material.

[0123] These molecules accumulate in atherosclerosis and mediate harmfuleffects (e.g., cholesterol, oxidized cholesterol, and other material,such as lysophospholipids) or in aging (e.g., sphingomyelin). Forexample, oxidized lipids, particularly sterols, alter many peripheraltissue functions, including stimulating calcification by arterial cellsin atherosclerosis & stimulating endothelial plasminogen activatorinhibitor-1 release by endothelial cells; other oxidized lipid productsinclude lysophospholipids that stimulate endothelial expression ofadhesion molecules that attract macrophages into lesions, andsphingomyelin accumulates in some cell-culture models of aging and, withcholesterol, may account for some of the cellular changes. Otherchanged, such as oxidation, may also mediate or accelerate aging. Manyof these molecules have been shown to be picked up by liposomes in vitro(e.g., cholesterol, sphingomyelin, & probably oxidized cholesterol) andmany by HDL (cholesterol, oxidized cholesterol by liposomes) but it islikely that they pick up these other molecules as well. In terms oftotal mass, however, the bulk of the acquired material is unesterifiedcholesterol, with proteins in second place. Alternatively, by acquiringunesterified cholesterol, the liposomes may reduce the amount ofoxidized cholesterol that develops, because there will be less startingmaterial.

[0124] The effective periods of time described herein should not beinterpreted to exclude very long courses of treatment, lasting years,for example. Nor should it exclude repeated courses of treatmentseparated by weeks, months, or years.

[0125] Side effects include overload of the liver with cholesterol orother materials acquired by the liposomes; with subsequent alterationsin hepatic function, such as suppression of LDL receptors, stimulationof intrahepatic cholesterol esterification, stimulation of intrahepaticcholesterol esterification, stimulation of hepatic secretion ofatherogenic lipoproteins that contain apolipoprotein-B, and impaireduptake of atherogenic lipoproteins by the liver from plasma.

[0126] As used herein the word, “endogenous” indicates that the HDLarises from within the body, and is not itself administered. HDL andrelated acceptors can, however, be administered.

[0127] The data indicates another difference between large and smallliposomes in vivo. Before injection, the liposomes that are used in ourexperiments were essentially electrically neutral, indicated by afailure to migrate rapidly through a gel of agarose when an electricfield is applied. (This does not imply that charged liposomes or otherparticles could not be used. The small liposomes pick up proteins andother material, and become electrically charged: they now rapidlymigrate through agarose gels when an electric field is applied. Agarosegels of plasma samples we had stored from the three groups of rabbitswere run. The small liposomes became more mobile LDL in these gels. Thelarge liposomes were substantially less mobile, indicating a lowercharge density, reflecting a lower protein content.

[0128] Two explanations for the difference between large and smallliposomes exist: 1) small ones penetrate through hepatic endothelialfenestrae while large ones do not (thus, large ones go to Kupffer cellsand small ones go to hepatic parenchymal cells and cause problems); 2)large liposomes are known to be cleared by the liver somewhat moreslowly than are small liposomes (the reason is not known), and so maynot overwhelm the liver as easily. The data on charge density providesan explanation in part: less protein, therefore slower or alteredhepatic uptake.

[0129] The delivery of cholesterol to the liver by LUVs is actually moreefficient than by SUVs, per mg of phospholipid. One difference is thatthe delivery by LUVs is steady over a long period after the injection,whereas the delivery by SUVs peaks then falls.

[0130] Some of the composition described herein include eggphosphatidylcholine; synthetic phosphatidylcholines that are notcrystalline at body temperature (e.g., they contain at least one doublebond) yet are resistant to oxidation (e.g., they do not have many doublebonds, such as 1-palmitoyl, 2-oleyl phosphatidylcholine, abbreviatedPOPC); other natural or synthetic phospholipids alone or in mixtures;any of the preceding supplemented or replaced with hydrophobic oramphipathic material that still allows a liposomal or micellarstructure. An extruder is certainly not the only conceivable method formaking large liposomes or even particularly LUVs. Other methods known topractioners in the field are available or can be adapted to make largeliposomes in general and LUVs in particular.

[0131] As used herein, a dose includes from 10 to 1600 mg ofphospholipid, in the form of large liposomes, per kg of body weight.Other acceptable rates described herein can be determined empirically bythe response of plasma LDL concentrations.

[0132] Where there is a change in membrane composition, as well asfunction, one can use an assay of membrane composition or an assay oftissue composition. Compositional assays should include lipids,proteins, and other components.

[0133] HDL can pick up oxidized material, and HDL-associated enzymes mayinactivate oxidized material.

[0134] The separations in time will depend on the actual dose ofmaterial, its effects on hepatic cholesterol homeostasis, and whethercholesterol-lowering agents are being concurrently administered. Thus,for doses of about 300 mg of small liposomes per kg of body weight,slight disruptions will occur after even a single dose, and singleadministrations of higher doses may cause even more disruptions.Exemplary separations in time include one day to one month, but theprecise schedules would have to be determined by monitoring hepaticcholesterol metabolism and plasma levels od LDL and other atherogeniclipoproteins.

[0135] The major macrophages that would be involved in liposomalclearance would be Kupffer cells in the liver and macrophages in thebone marrow or spleen. The catabolism here would be the so-calledalternative pathway for initiating the conversion of cholesterol intobile acids (macrophages are known to have at least onecholesterol-catabolizing enzyme), or would be transfer of sterol(enzymatically altered or not) to other cells, such as hepaticparenchymal cells that would then dispose of the molecules.

[0136] The methods described herein also control effects of cellularaging.

[0137] The invention includes means for assessing the efficacy ofliposomal therapy by performing assays of oxidation in vitro and invivo, assays of oxidative susceptibility of plasma components, andassays of the ability of altered HDL to inhibit oxidation (by bindingoxidative products and/or through its paroxinase or other anti-oxidantcomponents), and the ability of HDL or plasma or serum or blood tomobilize cholesterol and other exchangeable material.

[0138] Large liposomes may cause the mobilization of some material thatis trapped between cells as well (this is the extracellular space). Thisextracellular material causes problems a) when it contacts cells orplatelets, altering their function and b) by simply taking up space.

[0139] Estimate rates of cholesterol mobilization can be empiricallydetermined. It is appreciated that the kinetics of liposomal clearanceis different in different species (the t_(½) of LUVs in mice is about 8h, but in rabbits it is about 24th, and in humans it is longer). Thus,rates calculated may vary from species to species. Based on my data oninjection of 300 mg of SUVs into rabbits, the peak rate of liposomalcholesterol removal from plasma was between 3 h and 6 h after theinjection. At that point, the liposomes had raised plasma unesterifiedcholesterol by just over 2 mmol/L; assuming a total plasma volume of 90mL in a 3-kg rabbit, the total liposomal cholesterol at that point was180 umoles; the t_(½) for SUVs in these rabbits was about 20 h, soroughly 10% is removed in 3 h; thus, the peak rate of liposomalcholesterol removal was about 2 umoles/h/kg, and this caused asubsequent rise in plasma cholesteryl ester concentrations. Notice thatat other time periods after the injection, the rate of liposomalcholesterol removal from plasma was less. Note also that the liver isthe predominant organ for clearance, but not the sole organ forclearance.

[0140] It has been calculated that a single injection of 300 mg LUVs/kginto 20-22-g mice mobilized about 2400 nmoles of cholesterol in thefirst 24 h after injection. In contrast to the data with SUVs inrabbits, the mobilization of cholesterol during the first 24 h in themice injected with LUVs was quite steady. This calculates to about 4.7μmoles/h/kg over this first 24-h period, which is actually more than theabove figure of 2 μmoles/h/kg, which was a peak rate. It is not faircomparison, because the clearance of LUVs in mice is three times as fastas in rabbits. If we take 4.7 divided by 3, we get 1.6 umoles/h/kg,which is less than 2, but these are imperfect estimates. Human rates canbe empirically determined. It is clear, however, that LUVs deliver theircholesterol at a steady rate, whereas SUVs make a brief, rapid push oflipid into the liver.

[0141] At body temperature, the most desirable liposomes are fluidwithin the confines of the bilayer, which is called the liquidcrystalline state. Less desirable are liposomes in the gel state, whichis less fluid.

[0142] It is understood that unesterified cholesterol stimulatesmacrophages to express more tissue factor, a substance known to provokeblood clots. This explains the presence of abundant tissue factor inrupture-prone plaques, which, when they rupture, expose tissue factor toplasma and provoke a clot that can occlude the vessel, causing a heartattack. This would be another example of an abnormal cellular functionthat may be reversed by removal of cholesterol by liposomes.

[0143] Several human conditions are characterized by distinctive lipidcompositions of tissues, cells, membranes and/or extracellular regions.For example, in atherosclerosis, cholesterol (unesterified, esterified,and oxidized forms) and other lipids accumulated in cells and inextracellular areas of the arterial wall and elsewhere. These lipidshave potentially harmful biologic effects, for example, by changingcellular functions and by narrowing the vessel lumen, obstructing theflow of blood. Removal of the lipids would provide numerous, substantialbenefits. Moreover, cells, membranes, tissues and extracellularstructures would benefit from composition and alteration that includeincreasing resistance to oxidation and oxidative damages, such as byincreasing the content and types of anti-oxidants, removing oxidizedmaterial, and increasing the content of material that is resistant tooxidation. In aging, cells have been shown to accumulate sphingomyelinand cholesterol, which alter cellular functions. These functions can berestored in vitro by removal of these lipids and replacement withphospholipid from liposomes. A major obstacle to performing similarlipid alterations in vivo has been disposition of the lipids mobilizedfrom tissues, cells, extracellular areas, and membranes. Natural (e.g.,high-density lipoproteins) and synthetic (e.g., small liposomes)particles that could mobilize peripheral tissue lipids have asubstantial disadvantage: they delivery their lipids to the liver in amanner that disturbs hepatic cholesterol homeostasis, resulting inelevations in plasma concentrations of harmful lipoproteins, such aslow-density lipoprotein (LDL), a major atherogenic lipoprotein.

[0144] The invention described herein provides methods and compositionsrelated to the “reverse” transport of cholesterol and other materialsand compounds from peripheral tissues to the liver in vivo whilecontrolling plasma LDL concentration.

[0145] Agarose gel electrophoreses of plasma samples from the last a setof rabbits injected with LUVs, SUVs, or saline (these agarose gelsseparate particles by their charge, which is not the same from one typeof particle to another) were performed. Freshly made SUVs migrate veryslowly through agarose, which indicates that freshly made liposomes havevery little charge. After injection into animals or after co-incubationwith plasma or lipoproteins, SUVs pick up proteins from lipoproteins.These proteins give more charge to the SUVs and substantially enhancetheir migration through agarose gels. SUVs after exposure to plasmamigrate faster through these gels than does LDL.

[0146] The gels showed a substantial difference between LUVs and SUVs.As expected, the SUVs migrated ahead of LDL in these gels. The LUVs,however, migrated almost exactly where freshly made, protein-freeliposomes migrate. This result indicates that LUVs, unlike SUVs, do notreadily pick up proteins from circulating lipoproteins.

[0147] There is a direct verification of this difference between theliposomes. Human HDL (which has most of the proteins that liposomes pickup) was incubated with either LUVs or SUVs, then the liposomes werereisolated, and assayed their protein-to-phospholipid ratios. Per amountof liposomal phospholipid, the SUVs picked up about 40 times as muchprotein as did the LUVs. This difference appears to arise because of thedifference in surface curvature: SUVs are smaller, so their surface ismore tightly curved, thus under greater strain, proteins can more easilyinsert.

[0148] There are three most likely metabolic effects of the differencein protein uptake between the two types of liposomes are as follows:

[0149] 1. VLDL has two metabolic fates: it can be removed from plasmabefore it is fully converted to LDL by lipolytic enzymes, or it can befully converted into circulating LDL. SUVs strip apoE off VLDL, therebyslowing its clearance from plasma and favoring its conversion to LDL. Incontrast, LUVs leave apoE on VLDL, and so LDL concentrations in plasmawould not rise.

[0150] 2. Absorbed apoproteins might play a role in directing liposomesto different hepatic metabolic pools.

[0151] Here are some ways to assay effect on oxidation in vivo: CatellaF, Reilly M P, Delanty N, Lawson J A, Moran N, Meagher E, FitzGerald GA. Physiological formation of 8-epi-PGF2 alpha in vivo is not affectedby cyclooxygenase inhibition. Adv Prostaglandin Thromboxane Leukot Res.23:233-236, 1995. These authors describes 8-epi-PGF₂alpha, which is anend-product of lipid oxidation. This molecule can be used, they suggest,as a measure of lipid oxidative flux in an animal. It is superior toother commonly used measure of oxidation in vivo, such as anti-oxidantlevels (which are affected by diet), thiobarituric acid reactivesubstances (some sugars interfere with this assay), and short-livedoxidative intermediates (these do not indicate total flux of materialbeing oxidized). Administration of LUVs, by removing oxidized lipidsfrom the periphery, would might lower total oxidative flux in vivo, and8-epi-PGF₂alpha would be a suitable way to measure this; Cadet J,Ravanat J L, Buchko G W, Yeo H C, Ames B N. Singlet oxygen DNA damage:chromatographic and mass spectrometric analysis of damage products.Methods Enzymol. 234:79-88, 1994. they describe 8-oxo-2′-deoxyguanosine,which is an endproduct of DNA oxidation. As above, this molecule can beused as a measure of DNA oxidative flux in an animal. Administration ofLUVs would lower DNA oxidative flux in vivo, and this is a suitable wayto measure this; and, Xia E, Rao G, Van Remmen H, Heydari AR, RichardsonA. Activities of antioxidant enzymes in various tissues of male Fischer344 rats are altered by food restriction. J Nutr. 125(2):195-201, 1995.Antioxidant enzymes in tissues were measured, to indicate de-oxidantcapacity. LUVs help this. Anti-oxidant levels (vitamin E, ascorbate,urate); oxidized and reduced glutathione; and many other measures can beused to assess peripheral oxidation and oxidative damage. Again, theseand other measures would be coupled with LUV administration, to assessefficacy of the therapy.

[0152] Other particles that mimic there properties of large liposomeswill act similarly, to mobilize peripheral lipids and other exchangeablematerials, and deliver exchangeable materials, while avoiding harmfuldisruptions in hepatic cholesterol homeostasis. For example, these wouldinclude emulsion particles that are two large to penetrate hepaticendothelial fenestrae, of a composition and structure that is taken upby the liver slowly, and/or a composition and structure that does notreadily acquire specific endogenous proteins. Such emulsions could bemade with or without proteins, and could be made from phospholipid and aneutral lipid, such as triglycerides or another neutral lipid.

[0153] While only a few, preferred embodiments of the invention havebeen described hereinabove, those of ordinary skill in the art willrecognize that the embodiment may be modified and altered withoutdeparting from the central spirit and scope of the invention. Thus, thepreferred embodiment described hereinabove is to be considered in allrespects as illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are intended to be embraced herein.

I claim:
 1. A pharmaceutical composition consisting essentially of:large liposomes comprised of phospholipids substantially free of sterol,whereby said composition forces the reverse transport of cholesterolfrom peripheral tissues to the liver in vivo.
 2. A method of treatingatherosclerosis in a subject comprising the step of administering aliposome composition to said subject, said liposome composition selectedfrom the group consisting of unilamellar liposomes and multilamellarliposomes, said liposomes having an average diameter of about 50-150nanometers, in which LDL levels in said subject do not increase.
 3. Amethod of controlling cholesterol metabolism in hepatic parenchymalcells in a subject in vivo through cell-cell communication from Kupffercells to said parenchymal cells, comprising the step of administering aliposome composition to said subject, said liposome composition selectedfrom the group consisting of large unilamellar liposomes and largemultilamellar liposomes, said liposomes having an average diameter ofabout 50-150 nanometers, in which LDL levels in said subject do notincrease.
 4. The method in accordance with claim 3 in which the liposomecomposition is given periodically.
 5. The method in accordance withclaim 3 in which the liposome composition is given more than once. 6.The method in accordance with claim 3 in which the liposomes havediameters larger than about 50 nm.
 7. The method in accordance withclaim 3 in which the liposomes have diameters larger than about 80 nm.8. The method in accordance with claim 3 in which the liposomes havediameters larger than about 100 nm.
 9. The method in accordance withclaim 3 in which administration is selected from the group of parenteraladministration, intravenous administration, intra-arterialadministration, intramuscular administration, subcutaneousadministration, transdermal administration, intraperitonealadministration, intrathecal administration, via lymphatics,intravascular administration, including administration into capillariesand arteriovenous shunts, rectal administration, administration via achronically indwelling catheter, and administration via an acutelyplaced catheter.
 10. The method in accordance with claim 3 in whichabout 10 to about 1600 mg/kg/dose of said liposome composition isadministered.
 11. The method in accordance with claim 3 in which theliposome composition is given in repeated doses.
 12. The method inaccordance with claim 3 in which the liposomes are phospholipidssubstantially free of sterol and in the range of about 50-150 nm inapproximate diameter.
 13. The method in accordance with claim 3 in whichthe liposomes are phospholipids substantially free of sterol.
 14. Themethod in accordance with claim 3 in which the liposomes arephospholipids selected from the group consisting of phosphatidylcholine, phosphatidyl glycerol, palmitoyl-oleoyl phosphatidyl choline,combinations thereof, and derivatives thereof.